1. Field of the Invention
The present invention relates to a hepatitis C virus (HCV), more particularly, is related to a hepatitis C surrogate virus which comprises poliovirus genome, HCV protease and its target site, and is suitable for testing efficacy of anti-HCV drugs and screening the same, a recombinant gene and a use thereof.
2. Description of the Related Arts
There are known some types of viruses which cause viral hepatitis. Hepatitis A virus is an RNA virus having a diameter of 27 mn and causing epidemic hepatitis through oral infection. Hepatitis B virus is a DNA virus having a diameter of 42 mn and causing hepatitis through blood infection. A hepatitis virus which does not belong to any of the above types was called non-A, non-B hepatitis virus.
Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B hepatitis (Alter et al, N. Engl. J. Med. 321, 1494-1500, 1989; Choo et al., Science, 244, 359-362, 1989; Kuo et al., Science 244, 362-364, 1989). This virus has been implicated in liver cirrhosis and hepatocellular carcinoma (Bruix et al., Lancet ii, 1004-1006, 1989; Saito et al., Proc. Natl. Acad. Sci. USA, 87, 6547-6549, 1990). At present, xcex1-interferon is widely used for treating HCV patients. However, only about half of the patients respond to xcex1-interferon, and about half of the responders suffer from a recurrence of the virus (Hino et al., C. J. Med. Virol., 42, 299-305, 1994; Tsubota et al., Hepatology, 19, 1088-1094, 1994). Development of other anti-HCV drugs is, therefore, necessary.
In order to develop the anti-HCV drugs, it is essential to set up HCV cultivation system to test the efficacy of the drugs. However, in vitro cultivation systems available to date are inefficient and technically difficult to manipulate (Yoo et al., J. Virol., 69, 32-38, 1995; Beach et al., Viral Hepatitis Research Foundation of Japan, 286, 67, 1993; Shimizu et al., Proc. Natl. Acad. Sci. USA, 89, 5477-5481, 1992). Furthermore, HCV titer is very low even in sera of hepatitis patients due to its nature of low yield. Accordingly, it is very difficult to screen or test for efficacy of antiviral drugs using HCV cultivation. Therefore, the surrogate virus will be very useful for studying HCV genes in virus replication and testing antiviral efficacy of drugs affecting HCV-encoded enzymes. Namely, the development of the surrogate virus for anti-HCV drugs is urgent.
Accordingly, the present invention is intended to overcome the above-mentioned disadvantage in the development of anti-HCV drugs and provide proper surrogate viruses for HCV suitable for the investigation of antiviral efficacy of drugs and proliferable in the HCV cultivation system.
An embodiment of the present invention is a recombinant gene coding hepatitis C surrogate virus comprising a virus gene coding picornavirus, a protease gene coding hepatitis C virus protease NS3 which is in an open reading frame (ORF) of said picornavirus, and a target gene coding target sites of said hepatitis C virus protease NS3 which is in said open reading frame of said picornavirus.
Among the picornaviruses, the poliovirus is preferred.
The target site is preferred to be selected from the group consisting of NS5A/5B, NS4A/4B and NS4B/5A.
The hepatitis C surrogate virus protease NS3 is preferred to cleave the peptide bond between cysteine of P1 and glycine of P1xe2x80x2as a target site.
In another aspect, the present invention provides a screening method of anti hepatitis C virus drugs comprising the step of detecting a material which inhibits surrogate virus proliferation expressed by the recombinant gene for coding hepatitis C surrogate virus.
In another aspect, the present invention provides a measuring method of anti-hepatitis C virus drugs activity comprising the steps of proliferating the surrogate virus expressed by the recombinant gene coding hepatitis C surrogate virus in the presence of anti-hepatitis C virus agents, and measuring the proliferating ability of the hepatitis C surrogate virus.
The present invention, in another aspect, provides a hepatitis C surrogate virus comprising the recombinant gene for coding hepatitis C surrogate virus.
The poliovirus""s versatility is used in developing surrogate virus suitable for investigation of HCV protease activity.
HCV belongs to the family Flaviviridae whose members are enveloped viruses having a positive-sense RNA genome (Francki et al., Arch. Virol 2(Sullo.), 223, 1991; Inchauspe et al., Proc. Natl. Acad, Sci., 88, 10292-10296, 1991; Miller et al., Proc. Natl. Acad. Sci., 87, 2957-2061, 1990; Takamizawa et al., J. Virol. 65, 1105-1113, 1991). The RNA encodes a polyprotein (xe2x88x923010 amino acids) with the following gene order: 5xe2x80x2-Cxe2x80x2E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3xe2x80x2. During and/or after synthesis, the polyprotein is processed into functional proteins by host- and virus-encoded proteases. Core protein (C) and envelope proteins (E1 and E2) are believed to compose the structural elements of the virion particle. The balance of the processed proteins is believed to function in replication processes of the virus inside the host cells. The signal peptidase on the endoplasmic reticulum is responsible for the generation of the N-terminus of E1, E2, and possibly NS2. It has been suggested that the NS2/NS3 junction is cleaved by an HCV-encoded metalloprotease, which resides between the C-terminal portion of NS2 and the N-terminal region of NS3. The cleavage of the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites depends on the activity of the serine protease NS3 (Bortenschlager et al., J. Virol. 68, 5045-5055, 1993; Eckart et al., Biochem, Biophys. Res. Commun. 192, 399-406, 1993; Grakoui et al., J. Virol. 67, 2832-2843, 1993; Han et al., J. Gen. Virol.76, 985-993, 1995; Hijikata et al., J. Virol. 67, 4665-4675, 1993; Tomei et al., J. Virol. 67, 4017-4026, 1993). HCV protease NS3 is a good putative therapeutic target molecule for the development of anti-HCV drugs, since its activity is considered essential for viral proliferation. Protease activity of NS3 resides in the N-terminal part of NS3 (Bartenschlager et al., J. Virol. 67, 3835-3844, 1994; Han et al., J. Gen Virol. 76, 985-993, 1995; Lin et al., J. Virol. 68, 8147-8157, 1994), and NS4A enhances the protease activity of NS3 (Bartenschlager et al., J. Virol. 68, 3835-3844, 1994; Failla et al., J. Virol. 68, 3753-3760, 1994; Hahm et al., J. Virol. 69, 2534-2539, 1995; Lin et al., J. Virol. 69, 4373-4380, 1995; Tanji et al., J. Virol. 69, 1575-1581, 1995). Characterization of the NS3 protease using known protease inhibitors has revealed that NS3 is a chymotrypsin-like protease (Hahm et al., J. Virol. 69, 2534-2539, 1995).
The poliovirus is the prototype of picornaviruses. The polioviral genome is composed of a positive-sense RNA molecule that encodes a single open reading frame (ORF) (Kitamura et al., Nature (London) 291, 547-553, 1981; Racaniello et al., Proc. Natl. Acd. Sci. USA 78, 4887-4891, 1981). Upon infection, the genomic RNA is translated into a large precursor polyprotein via the internal ribosomal entry site (IRES) in the 5xe2x80x2-nontraslated region of the RNA (Jang et al., J. Virol. 62, 2636-2643, 1988; Pelletier et al., Nature (London) 334, 320-325, 1988; Pelletiet et al., J. Virol. 63, 441-444, 1989). The polyprotein, in turn, is processed into the mature viral structural and nonstructural proteins by the 2A, 3C, and 3CD proteases (Harris et al., Semin. Virol. 1, 323-333, 1991; Lawson and Semler., in xe2x80x9cCurrent Topics in Microbiology and Immunologyxe2x80x9d (V.R. Racaniello, Ed.), 161, 49-87, 1990). Poliovirus is one of the best studied viruses and is relatively easy to manipulate and cultivate. Production of infectious poliovirus from cDNA clones has been practiced for a decade to study functions of poliovirus proteins (Molla et al., Science 254, 1647-1651, 1991; Van der Werf et al., Proc. Natl. Acad. Sci. USA 83, 2330-2334, 1986) or for use of the poliovirus as a vector in delivering foreign genes into host cells (Alexander et al., Proc. Natl. Acad. Sci. USA 91, 1406-1410, 1994; Andino et al., Science 265, 1448-1451, 1994; Lu et al., J. Virol. 69, 4797-4806, 1995)
Inventors generated a hybrid poliovirus which requires the activity of HCV protease NS3 for its proliferation, on the basis of the characteristics of HCV and poliovirus. The hybrid virus is composed of poliovirus and HCV. The proteins necessary for the proliferation and infection into other cells are produced by poliovirus gene for the hybrid virus. The poliovirus gene is preceded by HCV protease and the sites cleaved by the protease to construct this hybrid virus which requires HCV protease NS3 activity for viability. This virus is composed, sequentially, HCV NS3 protease domain plus its target site, and open reading frame of poliovirus which is necessary for virus replication and virus particle formation. The activity of the enzyme NS3 protease is necessary to survive the hybrid virus.
The hybrid virus is proliferable in the cultivation system to settle the conventional problem which HCV cannot be proliferated in vitro cultivation systems. Therefore, the hybrid virus enables to develop anti-HCV drugs based on protease inhibitor easily and precisely.